There are five subtypes of dopamine receptors (DRs), classified into two primary categories, D1-like and D2-like. The D1-like family consists of the D1 and D5 dopamine receptors, whereas, D2-like family consists of the D2, D3, and D4 dopamine receptors. The D1 dopamine receptor (D1R may be linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition. Agents that enhance D1R signaling in the prefrontal cortex may be useful in the treatment of cognitive decline in schizophrenia, Alzheimer's disease and other dementias/age-related disorders. In FY18, we continued characterizing two D1 positive allosteric modulator (PAM) scaffolds, MLS1082 and MLS6585, previously identified in a high throughput screen. MLS1082 and MLS6585 potentiate dopamine (DA)-stimulated G-protein and beta-arrestin-mediated D1R signaling, increasing the potency of DA for stimulating cAMP accumulation and for beta-arrestin recruitment. Further, the two compounds potentiate the affinity of DA to bind to the D1R. Importantly, they show no ability to potentiate signaling by the D2-like receptors. Experiments using maximally effective concentrations of MLS1082 and MLS6585 in combination were used to determine if the compounds act at separate or similar binding sites. The combination of MLS1082 + MLS6585 was additive in potentiating DA's potency for stimulating beta-arrestin recruitment and cAMP accumulation, suggesting the two compounds act at separate sites. Repeating these combination experiments with Compound B, a known D1R PAM, showed additive activity with MLS6585, but not MLS1082, further suggesting that there are separate PAM binding sites in the D1R. A point mutation (R130Q) in the D1R was found to abrogate MLS1082 PAM activity without affecting that of MLS6585, suggesting this residue may be involved in the binding/activity of MLS1082, but not that of MLS6585. Together, MLS1082 and MLS6585 may serve as important tool compounds for the characterization of diverse allosteric sites on the D1R as well as the development of optimized lead compounds for therapeutic use. Despite its clinical importance in the treatment of a number of neuropsychiatric disorders, such as Parkinson's disease and schizophrenia, there are few compounds that are highly selective for the D2 dopamine receptor (D2R). Most compounds with activity at the D2R also exhibit significant affinity for the D3 or D4 receptors (D3R or D4R), or other G protein-coupled receptors (GPCRs). In FY18, we continued optimizing a highly selective antagonist for the D2R that we previously identified via a high-throughput screen - ML321. In a functional profiling screen of 168 different GPCRs, ML321 shows relatively little activity beyond inhibition of the D2R, and to a lesser extent the D3R, demonstrating exceptional GPCR selectivity. PET imaging studies in non-human primates demonstrate that ML321 can penetrate the CNS and occupy the D2R in a dose- dependent manner. Behavioral paradigms in rats demonstrate that that ML321 can selectivity antagonize a D2R-mediated response (hypothermia) while not affecting a D3R-mediated response (yawning) using the same dose of drug, thus demonstrating good in vivo selectivity. We also investigated the effects of ML321 in animal models that are predictive of antipsychotic efficacy in humans. We found that ML321 can attenuate both PCP and amphetamine-induced locomotor activity and pre-pulse inhibition (PPI) in a dose-dependent manner. ML321 also attenuates the hyperactivity seen in DA transporter (DAT) knockout mice. Importantly, using doses that are maximally effective in the locomotor and PPI studies, ML321 promotes little catalepsy compared with the non-selective antipsychotic haloperidol. These latter observations suggest that ML321 may produce fewer extrapyramidal motor side-effects (EPS), a common problem with FDA-approved antipsychotics. Overall, these results suggest that the ML321 scaffold can serve as a lead compound for the development of an improved therapeutic with greatly reduced side-effects for treating schizophrenia and other psychotic syndromes. The D2R signals through a variety of second messenger pathways making it difficult to discern which of these are linked to specific effects of D2R-targeted drugs; however, this complexity provides a unique opportunity to develop pathway-selective therapeutics. Structure-activity analyses using analogs derived from our previously described D2R G protein-biased agonist, ML1547, coupled with molecular modeling led to a structural model for biased signaling that entails a hydrophobic binding pocket formed by amino acid residues at the interface between the fifth transmembrane segment (TM5) and the second extracellular loop of the D2R (I184, V190 and F189). In FY18, we constructed point mutations at I184, V190 and F189 of the D2R, and at the aligned residues for F189 (i.e., position 5.38) within the D3R, D4R, and B2R, and studied their effects on G protein-mediated signaling and beta-arrestin recruitment. The D2R point mutations I184A and V190A produced a small change in the potency of DA for stimulating beta-arrestin recruitment or G protein activation. Strikingly, the F189A mutation ablated the ability of dopamine and other D2R agonists to recruit beta-arrestin while G protein-signaling efficacy was maintained. In addition, we found that mutating the D3R, D4R, and B2R at position 5.38 to Ala resulted in parallel findings (i.e., loss of agonist-stimulated beta-arrestin recruitment, but minimal effects on G protein-mediated signaling). These data demonstrate that the D2R F189A mutant, and similarly mutated D3R, D4R, and B2R, are highly biased towards G protein-mediated signaling and suggest that the presence of a F/Y residue at this position is important for stabilizing an activated state of the receptor for recruiting beta-arrestin. Conformational changes propagated through TM5 might thus act as a molecular switch for receptor signaling via beta-arrestin recruitment. These results may have implications for the design of novel signaling-biased compounds for the treatment of GPCR-related disorders In an effort to discover highly selective compounds for the D3R, our lab recently employed a high throughput screen of the NIH small molecule library. Hits were counter screened against the D2R to allow for the elucidation of compounds that activate the D3R without effects on the D2R. Orthogonal confirmation and counter-screens were also performed to obtain an initial assessment of selectivity and mechanisms of action. The most promising compound was chosen for chemical optimization and investigation of structure-activity relationships. 375 analogs were synthesized and screened in an effort to increase both affinity and selectivity of the scaffold. The lead compound identified through this process, ML417, acts as a full agonist at the D3R with nM potency while having minimal effects on D2R-mediated signaling. Importantly, the compound also exhibits potent and selective agonist activity in D3R G protein-mediated signaling responses. ML417 was further assessed for receptor cross-reactivity using multiple receptor panels and was found to have limited liability for off-target interactions with other GPCRs, thus demonstrating extreme selectivity for the D3R. Molecular dynamic simulations determined that ML417 binds to the D3R in a unique confirmation leading to its unique selectivity profile. As D3R-preferring agonists show promise as neuroprotective and neurorestorative agents, we conducted preliminary studies using ML417 in a neuroprotection assay and found that it displays neuroprotective properties. This highly selective and potent D3R agonist will prove useful as a research tool and may show utility as a therapeutic drug lead.